Antithrombin gene Arg197Stop mutation-associated venous sinus thrombosis in a Chinese family＊＊★

Ang Li, Tianhui Liu, Zhandong Liu, Jimei Li, Chunling Zhang, Jun Chen, Jinmei Sun, Yanfei Han, Lili Wang, Dexin Wang, Qiming Xue, Baoen Wang

1Department of Critical Care Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

2Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

3Department of Neurology, Health Care Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

4Department of Neurology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

lNTRODUCTlON

Patients with antithrombin (AT) deficiency are susceptible to thrombo-embolic diseases, particularly deep vein thrombosis of the lower limbs and pulmonary embolism[1].

AT functional deficiency is a strong risk factor for venous thrombosis. The gene encoding AT has been localized to chromosome 1q23-25[2]. The human AT gene is 13.5 kb in length and comprises seven exons and six introns[3].

Natural mutations of AT, primarily identified from families with a tendency toward thrombosis, have been collected in a database that has been published previously[4]and recently updated[5-6]. The identified mutations are grouped according to a proposed classification system[7]: type I and type II deficiencies are distinguished largely by the presence of variant protein in the latter group. Hereditary AT deficiency type I is caused by heterogeneous molecular defects with few partial or whole gene deletions and a majority of minor insertions, deletions or point mutations in exons 2-6 of the AT gene.

AT can combine with heparin and thrombin to prevent coagulation. There are two functional sites in AT: a serine protease reactive site located at Arg393-Ser394 and the other one related to Lys residues (position 125,107 and 136) and Arg residues (position 129 and 145). After AT genetic mutation, the amino acid sequence of the heparin binding site is changed, which can significantly reduce activity of AT and is likely to induce thrombosis[5,8].

There are rare clinical reports of cerebral venous sinus thrombosis (CVST) due to AT deficiency in a family pedigree. We are the first to report a case of CVST that was not relieved by heparin treatment and to carry out a study of the genetic associations of AT among the patient’s family members.

RESULTS

AT level assays in pedigree members with AT deficiency

The results obtained from functional and immunological AT assays are summarized in Table 1. Multiple analyses found that the AT antigen level in the propositus patient was obviously higher than that in other pedigree members. AT activities of the propositus, her brother, niece and daughter were all lower than those in normal controls (approximately 50%). Her mother and husband had normal AT levels and activities. Her father died from venous thrombosis of the lower limbs that was complicated by pulmonary embolism at the age of 60, but the AT level was not determined.

Genomic basis in pedigree members with AT deficiency

All seven exons, including splice junctions,of the gene from the propositus were amplified by PCR.Subsequent DNA sequencing revealed that only the exon 3B fragment differed from normal controls. A C to T substitution in one allele of the AT gene at position 6431,predicted to result in a nonsense mutation of an arginine(CGA) into a stop codon (TGA), was found. Next, we analyzed exon fragment 3B from her mother, husband,brother, niece and daughter. The propositus’ mother and husband were normal, while the other family members had the same nonsense mutation (Figure 1).

Table 1 Clinical characteristics and antithrombin levels in the propositus and her family members

Figure 1 DNA sequencing results of exon 3B of the antithrombin gene from the patient (A) and a control (B).Subsequent DNA sequencing revealed that only the exon 3B fragment differed from normal controls. Arrow shows a C to T substitution in one allele of the antithrombin gene at position 6431, predicted to result in a nonsense mutation of an arginine (CGA) into a stop codon (TGA).

The single strand conformation polymorphism patterns of the amplified PCR products of the AT 3B exon from the family confirmed that, with the exception of her mother and husband, the propositus and other members exhibited abnormal patterns due to the described point mutation (Figure 2).

Figure 2 Single-strand conformation polymorphism patterns of amplified PCR products of the antithrombin exon 3b from six family members. Samples were run on 80 g/L non-denaturing polyacrylamide gels in 1× TBE buffer for 8–12 hours at 40 V. Due to genetic mutation,bands with the same number of base pairs can adopt different conformations and show different resolution patterns by electrophoresis. Both of the bands in Figure 2 are 233 bp. Since the patient’s mother and husband do not have the mutation, their band type is different from those of other family members with the mutation.

The C to G mutation destroyed one of three Hae III restriction sites normally present in the exon 3B PCR fragment[9]. Normally, fragment 3B is cut into 4 fragments of 66, 34, 92 and 40 bp after Hae III digestion, whereas the mutant allele produces fragments of 66, 126 and 40 bp (Figure 3).

Figure 3 PCR products of antithrombin exon 3B from the patient and her family, digested by Hae III. Normal antithrombin exon 3B is cut into four fragments at 66, 34,92 and 40 bp after Hae III digestion, whereas the mutant allele is cut into fragments of 66, 126 and 40 bp. The control is a PCR product of undigested antithrombin exon 3b fragment from people without the mutation.

DlSCUSSlON

This is the first report of a point mutation C6490T causing clinically symptomatic type I AT deficiency in a Chinese family. A Japanese family and a Caucasian family with the same point mutation and symptomatic hereditary type I AT deficiency have been previously reported[9-10]. A single C to T point mutation at nucleotide position 5381,resulting in the nonsense mutation R129X, has been identified in eight families with type I AT deficiency. The rather frequent occurrence of this mutation could be explained by its presence in a CpG dinucleotide hotspot[9].

The presence of the R197X mutation, which also occurs in a CpG dinucleotide in Dutch, Japanese and Caucasian families with symptomatic hereditary AT deficiency type I,strongly suggests that the mutations have arisen independently, and not from a founder effect, as their identity by descent is very improbable.

In this study, diagnosis was verified by the clinical manifestations and radio-imaging results. Laboratory examinations revealed obviously decreased plasma AT activity and elevated antigen concentration. Genetic studies showed that the patient had a hereditary nonsense mutation(Arg197Stop) of the AT gene associated with AT deficiency.

The clinical diagnosis of CVST might have been due to hereditary AT deficiency. Other pedigree members with same genetic defect also showed reduced blood AT activities, though the AT levels were still normal. Thus, in the absence of any factors promoting a hyperaggregative state, a patient can still maintain a normal aggregation and fibrolytic equilibrium that does not result in thrombosis formation, despite the presence of genetic defects and protein functional abnormalities.

Underlying predisposing factors can be identified in up to 80% of CVST patients[11-12]. Numerous conditions can predispose one to CVST and frequently more than one factor will be found in an individual patient. Infective causes were responsible for only 8% of cases in recent series, and their prevalence has declined[13-14]. Among non-infective causes, systemic conditions such as connective tissue diseases, anemias, granulomatous and inflammatory bowel diseases, as well as malignancies,are most common[12-13]. Hereditary prothrombotic conditions such as factor V Leiden, deficiency of proteins C and S and AT, as well as prothrombin gene mutations may account for one-third to two-thirds of children and 10-15% of adults with CVST[15-17]. In the propositus patient, the chronic state of the antetibial ulceration might have activated a prothrombotic state. Because of the existence of her genetic defects, the patient could not antagonize the body’s thrombogenic factors, which caused intracranial venous sinus thrombosis, although patient had a compensatory increase in AT synthesis.

CVST presents with a wide spectrum of symptoms and signs, and sometimes the clinical manifestations are non-specific and subtle[11]. Clinically, a decompression procedure was frequently used on some obvious intracranial hypertensive patients[18]. Although the patient had typical imaging features of cerebral edema, mannitol treatment improved symptoms of restlessness and delirium, while two lumbar punctures showed significant lower intracranial pressure. This manifestation was rare compared with that in patients with venous sinus thrombosis from other causes. The pathogenesis may be associated with her genetic defect.

In summary, this CVST case exhibited brain edema due to chronic disturbance of venous back-flow, and the observed reduction in intracranial pressure might have been a compensatory mechanism to alleviate brain edema. Whether these features differentiated the specific characteristics of CVST from the AT deficiency should be further verified by advanced studies. However,the therapeutic treatment of this case indicates that dehydration treatment should be used in CVST cases with chronic, insidious onset and obvious brain edema, even when intracranial pressure is within normal limits or at lower levels.

In addition, anti-coagulation therapy remains one of the mainstay treatments for venous thrombosis[19-20]. We administered heparin for anti-coagulation before AT deficiency was verified, consistent with the treatment recommendations of the European Federation of Neurological Societies guidelines on the treatment of CVST thrombosis in adult patients[21]. However, it is not difficult to conclude from this study that CVST patients can also have AT deficiency when heparin treatment does not have any effect.

SUBJECTS AND METHODS

Design

A genetic association analyzing study.

Time and setting

The study was performed at the Beijing Friendship Hospital Affiliated to Capital Medical University, China, from June 2009 to July 2010.

Subjects

We collected data and DNA samples from 6 members of 3 generations of this pedigree for genetic and clinical studies. Figure 4 shows the pedigree distribution. Except for the patient’s father, who died from venous thrombosis of the lower limbs complicated by pulmonary embolism at the age of 60 years, the patient’s other family members did not have a history of thrombosis.

Figure 4 Family pedigree. I, II, III: Different generations of the pedigree; 1, 2, 3: different members in each generation of the pedigree; AT: antithrombin.

The propositus patient was a 40-year-old female admitted for a chronic ulcer of the left antetibial region that had been present for 4 months, and recurrent headache that had been present for 2 months and became aggravated in the 8 days prior to admission. She suffered from left leg deep vein thrombosis after childbirth and thrombectomy had been performed. She complained of recurrent headaches, mostly in bitemporal areas, that persisted for about 15 minutes per attack. The headaches had no relation to body posture but they were usually accompanied by agitation. No nausea, emesis or fever was noted.

She was able to perform her daily work activities at this time. Eight days before admission, when her headache became aggravated, it was first associated with nausea and severe emesis, and later also with urinary incontinence, restlessness and mental disturbance.

The patient was delirious upon physical examination and had slight nuchal rigidity, but no signs of cranial nerve or limb paralysis, no bilateral papilledema and no vision impairment. Pathological reflexes were positive bilaterally and urinary incontinence was observed. A head computer tomography scan showed higher signal intensity along the longitudinal fissure with marked brain edema. Magnetic resonance imaging of venous scan showed multiple filling defects on the superior sagittal,confluent, rectal and left transverse sinuses. Two con-secutive lumbar punctures during hospitalization showed cerebrospinal fluid pressures of 0.64 and 0.49 kPa.

Total cerebrospinal fluid protein concentration was 115 mg/dL. Other biomarkers were all within normal limits.

Blood biochemical markers were apparently normal, but blood coagulation coefficients showed marked reduction of AT-III. Antiphospholipid antibodies, lupus anticoagulants, protein C, and protein S activities were all normal.

CVST was considered as a clinical diagnosis and initial treatment included dehydration with mannitol as well as low-molecular heparin therapy, with no obvious effects.

After switching to warfarin treatment, the CVST symptoms were gradually alleviated, the antetibial ulceration healed and she was discharged. Imaging features are shown in Figure 5.

Figure 5 Brain images of the cerebral venous sinus in our thrombosis patient.

Methods

Blood sample collection from all members of the patient’s family

Blood samples were collected from the antecubital vein at 1: 10, with 0.11 mmol/L trisodium citrate. Plasma was prepared by centrifugation for 10 minutes at 2 000 × g at 10°C and stored at -70°C for further use[22]. Genomic DNA was isolated from peripheral blood leukocytes.

AT assays in all members of the patient’s family, as detected by the synthetic chromogenic substrate method

AT concentration in serum was manually measured by a immunofluorescence turbidity assay in accordance with the kit’s instructions (Shanghai Tai-yang Bio-products Co., China) using an ultra-violet spectrometer (JENWAY 6505, Dunmow, Essex, England). AT concentration standards were provided with the kit.

The AT activity level in plasma was measured using the synthetic chromogenic substrate method[23]. First,plasma was incubated with a known excess of thrombin in the presence of heparin. Residual thrombin was quantified based on its amidolytic action on the synthetic chromogenic substrate CBS 61.50 (pNA release measured at 405 nm). The reagents and equipment used were STA-Stachrom AT III kits and a Coagulation Analyzer(STA-R Evolution, Asnieres, France). AT activity standards were provided with the kit.

DNA sequence analysis of AT exons in all members of the patient’s family

All seven exons including splice junctions were amplified by PCR using the primer sets and PCR reaction conditions listed in Table 1. Genes were analyzed by direct sequencing[24].

Primer Sequences Product size (bp) Position AT1F: 5′ -ACA ACA CTG GGC TCT ACA C-3′509 71-579 R: 5′-ACT TCC TGC ACA GCA TCT T-3′AT2F: 5′-TAA CTT GGC ATT TTG TCT CC-3′418 2384-2801 R: 5′-CTG GGC AGA AGA CCT TG-3′AT3A F: 5′-AAA CCC ACC ACC ATT TTT T-3′289 5276-5564 R: 5′-AGG AAG AAC TCG GAG GTC AG-3′AT3B F: 5′-TTG AAT AGC ACA GGT GAG TAG G-3′233 6387-6619 R: 5′-GCT GAA GAG CAA GAG GAA GT-3′AT4F: 5′-TGT GAT TCT CTT CCA GGG C-3′442 7362-7803 R: 5′-GGT GGA GAA GGG AGG AAA C-3′AT5F: 5′-CAA CTT TCT CCC ATC TCA CAA-3′147 9757-9903 R: 5′-TTT CTG TAC CCT AAG AGA GTG G-3′AT6F: 5′-GTT TTA TTC CCA TGT GAC CTG-3′210 13217-13426 R: 5′-AAG AAC ATT TTA CTT AAC ACA AGG-3′

Single-strand conformation polymorphism analysis of PCR products in all members of the patient’s family

PCR products were separated by electrophoresis on 12 g/L agarose gels to check the specificity of each PCR reaction. The products were mixed with the same amount of formamide loading buffer (950 g/L formamide,10 mmol/L EDTA, and 0.5 g/L bromophenol blue, 0.5 g/L xylene cyanol), heated at 100°C for 5 minutes, and loaded onto 80 g/L non-denaturing polyacrylamide gels in 1× TBE buffer for 8-12 hours at 40 V. After electrophoresis, the gels were fixed, silver-stained, and finally,developed, photographed and analyzed with Quantity One software (BioRad, Hercules, CA, USA). The sample was considered normal if the band position was the same as that of the normal control, for example, the mother or husband. Otherwise, it was considered abnormal[9].

Restriction enzyme analysis of AT 3B PCR fragment mutations in all members of the patient’s family

PCR fragments (10 μL) of the involved exons from patients and normal controls were digested with 2 U of Hae III at 37°C for 2 hours in a total volume of 20 μL. After digestion, the products were applied to 20% polyacryla-mide gels and run at 100 V for 70 minutes in a BioRad Vertical Electrophoresis Unit (BioRad, Hercules, CA,USA). The products were visualized by Gelred staining[9].

Author contributions:Zhandong Liu was responsible for the study design, data collection and integration, fund charge. Ang Li, Tianhui Liu, Jimei Li, Chunling Zhang, Jun Chen, Jinmei Sun and Yanfei Han participated in data collection and integration.Lili Wang provided technical and material support. Dexin Wang was the research director. Qiming Xue was the article validator.Baoen Wang was responsible for the study design.

Conflicts of interest:None declared.

Funding:The study was financially supported by the National Natural Science Foundation of, China. No. 81041019, “The multi-directional studies of 5-hydroxy-tryptaminergic system in central fatigue: Biochemical, electrophysiological and MRS methods on clinical patients and experimental animals”; the National High-Technology Research and Development Program of China (863 Program), No. 2006AA02Z436, “The study of clinical diagnostic and biomedical criteria for evaluating sub-health population with chronic fatigue syndrome”.

Ethical approval:The study was approved by the Ethical Review Board of Beijing Friendship Hospital Affiliated to Capital Medical University, China.

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